Antimicrobial use of annexin a1 protein

Abstract

There is described Annexin A1 protein, and variants thereof, for use in the treatment of a microbial disorder.

Description

[0001] Antimicrobial Use of Annexin Al Protein

[0002] Field of the Invention

[0003] The present invention relates to Annexin Al protein as well as compositions comprising said protein or protein analogues for use as antimicrobial agents.

[0004] More particularly, the invention relates to Annexin Al protein; and protein analogues thereof, for use as antimicrobial agents, especially antibacterial agents.

[0005] Background to the Invention

[0006] Some of the most important antibiotics currently available are the beta-lactam antibiotics, which include penicillins, penems, carbapenems, cephalosporins, monobactams and sulfactams. Beta-lactam antibiotics inhibit cell wall biosynthesis by binding to proteins called penicillin-binding proteins (PBPs), which are essential for synthesis of peptidoglycan, the major component of the cell wall of Gramnegative and Gram-positive bacteria. However, the misuse and overuse of betalactam, as well as other antibiotics have led to a large and growing problem of antibiotic resistance.

[0007] Beta-lactam antibiotics are among the few classes that are effective against Gramnegative bacteria. Gram- negative bacteria have little peptidoglycan in their cell wall. Bacterial cells often have antigens in their cell wall, which allow the human body to recognise them. However, Gram-negative bacteria often have a capsule covering so that these antigens are hidden from the immune system and the immune system cannot target the infective bacteria. Furthermore, some gram-negative bacteria produce enzymes (such as carbapenemase and beta-lactamase active towards carbapenems and beta-lactams respectively) which are able to degrade antibiotics.

[0008] In addition, lipopolysaccharide, which is found in the outer cell wall of Gramnegative bacteria, can cause inflammation, which can be severe enough to trigger sepsis. This cell wall also stops antibiotics penetrating the bacteria, making Gramnegative infections more difficult to treat than Gram-positive infections. Gramnegative bacteria also use efflux pumps (transmembrane protein on the inner membrane and periplasmic protein and porin complex) which allow for the transport of antibiotics out of the cells. Some efflux pumps are able to transport more than one antibiotic class. Examples of Gram-negative bacteria include, Acinetobacter e.g. Acinetobacter baumannii, Pseudomonas e.g. Pseudomoaas aeruginosa, Enterobacteriaceae (e.g. Escherichia coli (E. colt), Klebsiella pneumoniae, Shigella spp., Salmonella spp.), Campylobacter spp., Neisseria e.g. Neisseria gonorrhoeae, Helicobacter e.g. Helicobacter pylori, Haemophilus e.g. Haemophilus influenzae, and Vibrio e.g. Vibrio cholerae.

[0009] Certain bacteria are associated with drug resistant or even multidrug resistant strains that are a cause of concern in antimicrobial resistance (AMR). For example, Acinetobacter baumannii - carbapenem-resistant, Pseudomonas aeruginosa - carbapenem-resistant, Enterobacteriaceae - carbapenem-resistant, 3 generation cephalosporin-resistant, Shigella spp. - fluoroquinolone-resistant, Salmonella spp. - fluoroquinolone-resistant, Campylobacter spp. - fluoroquinolone-resistant, Neisseria gonorrhoeae - 3 generation cephalosporin-resistant, fluoroquinolone-resistant, Helicobacter pylori - clarithromycin-resistant, Haemophilus influenzae - ampicillin- resistant.

[0010] Gram-positive bacteria are not usually found in the human body, which means that they are more easily recognised and targeted by the immune system. Furthermore, Gram-positive bacteria are generally more susceptible to beta-lactam antibiotics like penicillin.

[0011] However, Gram-positive bacteria can still acquire antibiotic resistance. Examples of Gram-positive bacteria include Staphylococcus e.g. Staphylococcus aureus, Enterococcus e.g. Enterococcus faecium, Streptococcus e.g. Streptococcus pneumoniae, and Clostridium e.g. Clostridium botulinum. Certain gram-positive bacteria are extremely important pathogens in both human and veterinary medicine, and drug and multidrug resistance is a cause for concern. Some examples are Staphylococcus aureus - vancomycin-resistant (VRSA), methicillin-resistant (MRSA), Enterococcus faecium - vancomycin-resistant (VREF), Streptococcus pneumoniae - penicillin-non-susceptible.

[0012] Staphylococci are major causes of serious healthcare associated infection (HAI). Of particular note are strains of Staphylococcus that have developed or obtained varying levels of resistance to antibiotics such as methicillin (meticillin). These difficult-to- treat organisms are commonly known as methicillin resistant Staphylococcus aureus (MRSA) and methicillin resistant Staphylococcus epidermidis (MRSE). Approximately 80% of S. epidermidis isolates from device-associated infections are methicillin resistant (MRSE) as well as being multi-resistant. Resistance to multiple antibiotics and the ability of S. epidermidis to form biofilms on inert surfaces exacerbate the challenges of treating infections caused by these organisms. Similarly, reports of methicillin-resistant S. aureus (MRSA) in animals have become more frequent in recent years.

[0013] Clostridia are multi-drug resistant Gram-positive bacteria that are becoming one of the most difficult to treat healthcare-associated infections. The use of broad-spectrum antibiotics reduces bacterial colonisation in the intestine, permitting the overgrowth of C. difficile. One of the key challenges in the treatment of C. difficile is the fact that it is able to form these highly resistant spores. As such, antibiotic treatment commonly results in the inhibition of the actively growing Clostridium cells but not the vegetative spores. The spores remain in the gut of a mammal after treatment and can then germinate resulting in a new C. difficile infection. Currently the antibiotics of choice for the treatment of C. difficile are metronidazole and then vancomycin if metronidazole is ineffective. However, neither of these drugs is able to inhibit the outgrowth of Clostridium spores, and as such the prevalence of relapses in C. difficile infection after treatment is estimated at around 55% of all cases.

[0014] Antibiotic or antimicrobial resistance and its association with serious infectious diseases has increased at alarming rates. On a global level, antimicrobial resistance was reported to have killed at least 1.27 million people worldwide in 2019, and was associated with nearly 5 million deaths. In particular, the increasing prevalence of resistance among nosocomial pathogens is causing alarm. In the USA more than 2 million nosocomial infections occur each year and it is estimated that about 50 to 60% such infections are caused by antimicrobial-resistant strains of bacteria. The high rate of resistance to commonly used antibacterial agents increases the morbidity, mortality, and costs associated with nosocomial infections. In the USA nosocomial infections are estimated cause or contribute to an excess of 77,000 deaths per year and cost annually approximately $5 to $10 billion. Further to nosocomial infections, the increased prevalence of drug-resistant bacteria difficult to treat with current antibiotics leads to an increased risk of complications carrying out multiple procedures such as surgeries, organ transplants and chemotherapy. For these procedures, effective antibiotics for the prevention and treatment of infections are vital.

[0015] Therefore, there is a need to provide a new and effective treatment for infections caused by Gram-negative and / or Gram-positive bacteria, such as, Acinetobacter e.g. Acinetobacter baumannii, Pseudomonas e.g. Pseudomonas aeruginosa, Enterobacteriaceae (e.g. Escherichia coli (E. colt), Klebsiella pneumoniae, Shigella spp., Salmonella spp.), Campylobacter spp., Neisseria e.g. Neisseria gonorrhoeae, Helicobacter e.g. Helicobacter pylori, Haemophilus e.g. Haemophilus influenzae, Vibrio e.g. Vibrio cholerae, Staphylococcus e.g. Staphylococcus aureus, Enterococcus e.g. Enterococcus faecium, Streptococcus e.g. Streptococcus pneumoniae, and Clostridium e.g. Clostridium botulinum. The list is inclusive of drug-resistant strains as provided above.

[0016] We have now surprisingly found that Annexin Al protein possesses antimicrobial properties. Annexin Al is a protein with a relative molecular mass of about 40 kDa. The protein is encoded by the ANXA1 gene in humans. It belongs to the Annexin family of Camdependent phospholipid-binding proteins.

[0017] Annexin proteins are involved in various biological activities including membrane trafficking, transmembrane channel activity, inhibition of phospholipase A2, coagulation inhibition, transduction of mitogen signals, and mediation of cell-matrix interaction, etc.

[0018] Humans are reported to have Annexin I, II, III, IV, V, VI, VII, VIII, IX, X, XI and XIII. Annexin Al protein has been found in amnion epithelial cells and chorion laeve trophoblast, and the syncytiotrophoblast cells of placenta. It is also present in the lungs, intestinal tract and bone marrow. It is expressed in hematopoietic cells such as monocytes, eosinophils and neutrophils. Annexin Al is also known to be detected in some cancer tissues as well.

[0019] Annexin Al is implicated in the regulation of a number of cellular functions, including cell signalling, secretory processes (e.g. hormonal secretion), prolactin release and prostaglandin formation. It is a critical component of the blood-brain barrier (BBB) and it plays a major protective role in the brain through the promotion of BBB integrity. Exogenous and endogenous Annexin Al counter-regulates the activities of innate immune cells, in particular, it promotes anti-inflammatory activity as well as extravasation and the generation of pro-inflammatory mediators, ensuring that a sufficient level of activation is reached but not exceeded. Overall, the role of Annexin Al in the body is diverse. It is involved with maintenance of a homeostatic environment, foetal development, the aging process and development of disease.

[0020] International Patent application No. WO 2022 / 038258 describes the use of Annexin Al and compositions comprising Annexin Al in the treatment of inflammatory conditions, such as, rheumatoid arthritis, stroke, myocardial infarction, septic shock, an inflammatory skin disorder, etc.

[0021] Korean Patent application No. KR 2018 / 0016243 describes separating at least one of the amniotic membranes, including the chorionic membrane and the epithelial cell layer, from the placenta, to obtain a membrane extract with antibiotic activity.

[0022] Summary of the Invention

[0023] Thus, according to a first aspect of the invention there is provided Annexin Al protein, and variants thereof, for use in the treatment of a microbial disorder.

[0024] The term “microbial disorder” as used herein shall include, for example, disorders caused by bacteria and other microbes, such as, parasites (e.g. malaria), viruses (e.g. HIV) and fungi (e.g. Candida). In a particular aspect of the invention the “microbial disorder” is a bacterial disorder, e.g. a disorder caused by Gram-negative bacteria or Gram-positive bacteria.

[0025] Thus, the Annexin Al protein of the present invention is antimicrobial. The term “antimicrobial” shall encompass an agent that kills microbes, i.e. microbicidal; and an agent that inhibits the growth of microbes, i.e. microbiostatic. In particular, the Annexin Al protein of the present invention is antibacterial, i.e. bactericidal and / or bacteriostatic. The Annexin Al protein of the present invention may possess antibacterial and anti-inflammatory properties.

[0026] In a further embodiment, the Annexin Al protein of the present invention may be used prophylactically as a means to prevent the development of an infection. Medicaments and / or methods for prophylactic use may be administered or applied to any person at risk of developing an infection, for example, people working in care homes, nursing homes, sports centres, community centres, shops, restaurants, cafes, nurseries and / or schools may require prophylactic treatments.

[0027] The Annexin Al protein herein described is commercially available, from numerous protein suppliers. Variants of modifications of Annexin Al can be purchased from custom protein synthesis suppliers.

[0028] The “variants” or “modifications” of Annexin Al protein include individual substitutions, deletions, or additions to a protein sequence which result in the substitution of an amino acid with a chemically similar or non-chemically similar amino acid or a non-amino acid moiety. Substitution tables providing functionally similar amino acids are well known in the art.

[0029] Variants of the Annexin Al protein herein described may possess substitutions, additions, deletions and other modifications, and so lead to derived proteins. Ordinarily, variants of the Annexin Al protein will possess at least about 50% identity (homology) to the reference to Annexin Al protein, and preferably, they will be at least about 80%, or at least about 85%, more preferably at least about 90%, even more preferably at least about 95% identical (homologous) to the Annexin Al protein.

[0030] The Annexin Al protein herein described, and variants thereof, can be administered to treat bacterial strains that exhibit resistance to beta-lactam antibiotics. The Annexin Al protein herein described can be used to treat infections caused by Gram-negative bacteria, including, but not limited to, Acinetobacter e.g. Acinetobacter baumannii, Pseudomonas e.g. Pseudonomas aeruginosa, Enterobacteriaceae (e.g. Escherichia coli (E. coli), Klebsiella pneumoniae, Shigella spp., Salmonella spp., Campylobacter spp., Proteus, Enterobacter, Serratia, and Citrobactef), Neisseria e.g. Neisseria gonorrhoeae, Helicobacter e.g. Helicobacter pylori, Haemophilus e.g. Haemophilus influenzae, Vibrio e.g. Vibrio cholerae, non-fermenting bacteria, including Burkholderia, Moraxella and Stenotrophomonas, as well as anaerobic bacteria, such as Bacteroides fragilis or Bacteroides thetaiotaomicron: or Gram-positive bacteria, including, but not limited to, Staphylococcus e.g. Staphylococcus aureus, Enterococcus e.g. Enterococcus faecium, Streptococcus e.g. Streptococcus pneumoniae, and Clostridium e.g. Clostridium botulinum. This list is inclusive of drug-resistant and multi-drug resistant strains. It is a particular aspect of the invention that the Annexin Al protein herein described, and variants thereof, can be used to treat infections caused by those pathogens appearing on the World Health Organisation (WHO) list of priority pathogens: WHO updates list of drug-resistant bacteria most threatening to human health. Specific pathogens which may be mentioned include, but shall not be limited to, Gram negative strains, such as, E. coli, Pseudomonas aeruginosa,' and Gram positive strains, such as, Streptococcus pneumoniae & Staphylococcus aureus. In another aspect, the invention provides a pharmaceutical composition comprising Annexin Al protein, and variants thereof, admixed with at least one pharmaceutically acceptable carrier or excipient, for use in the treatment of a microbial disorder.

[0031] In further embodiments the composition of the invention may comprise two or more such carriers or excipients.

[0032] In another aspect of the invention the pharmaceutical composition may include Annexin Al protein in combination with one or more additional antibiotics or other therapeutic agents. It will be understood by the person skilled in the art that the Annexin Al protein may be formulated and administered separately from the other antibiotic.

[0033] The one or more other antibiotic may be selected from one or more of the antibiotic classes aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins glycopeptides, lincosamides, lipopeptide, macrolides, monobactams, nitrofiirans, oxazolidinones, penicillins, polypeptides, quinolones, sulfonamides, tetracyclines and drugs against mycobacteria. The one or more other antibiotic may be a peptide fragment of Annexin Al as described in our co-pending application No. GB 2315799.3.

[0034] Examples of aminoglycosides include, but shall not be limited to, amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, paromomycin, streptomycin and spectinomycin. Examples of ansamycins include, but shall not be limited to, geldanamycin, herbimycin and rifaximin.

[0035] Examples of carbacephems include, but shall not be limited to, loracarbef.

[0036] Examples of carbapenems include, but shall not be limited to, ertapenem, doripenem, imipenem and meropenem.

[0037] Examples of cephalosporins include, but shall not be limited to, cefadroxil, cefazolin, cephradine, cephapirin, cephalothin, cefalexin, cefaclor, cefoxitin, cefotetan, cefamandole, cefmetazole, cefonicid, loracarbef, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, moxalactam, ceftriaxone, cefepime, ceftaroline fosamil and ceftobiprole.

[0038] Examples of glycopeptides include, but shall not be limited to, teicoplanin, vancomycin, telavancin, dalbavancin and oritavancin.

[0039] Examples of lincosamides include, but shall not be limited to, clindamycin and lincomycin.

[0040] Examples of lipopeptide include, but shall not be limited to, daptomycin. Examples of macrolides include, but shall not be limited to, azithromycin, clarithromycin, erythromycin, roxithromycin, telithromycin, spiramycin and fidaxomicin.

[0041] Examples of monobactams include, but shall not be limited to, aztreonam.

[0042] Examples of nitrofiirans include, but shall not be limited to, furazolidone and nitrofurantoin.

[0043] Examples of oxazolidinones include, but shall not be limited to, linezolid, posizolid, radezolid and torezolid.

[0044] Examples of penicillins include, but shall not be limited to, amoxicillin, ampicillin, azlocillin, dicloxacillin, flucioxacillin, mezlocillin, methicillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, temocillin and ticarcillin.

[0045] Examples of polypeptides include, but shall not be limited to, bacitracin, colistin and polymyxin b.

[0046] Examples of quinolones include, but shall not be limited to, ciprofloxacin, enoxacin, gatifloxacin, gemifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin and temafloxacin. Examples of sulphonamides include, but shall not be limited to, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprimsulfamethoxazole (co-trimoxazole) and sulfonamidochrysoidine.

[0047] Examples of tetracyclines include, but shall not be limited to, demeclocycline doxycyclcycline, metacycline, minocycline, oxytetracycline and tetracycline.

[0048] Examples of drugs against mycobacteria include, but shall not be limited to, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifampicin, rifabutin, rifapentine and streptomycin.

[0049] In another aspect, the invention provides a method for treating a subject having a microbial infection, which comprises administering to the subject in need thereof an antimicrobially effective amount of Annexin Al protein.

[0050] The method according to this aspect of the invention may comprise administering a pharmaceutical composition as herein described, i.e. optionally in combination with a pharmaceutically acceptable carrier. In certain embodiments, the subject is a mammal and in some embodiments, the subject is a human.

[0051] According to this aspect of the invention the method may comprise the treatment of a Gram-negative bacterial infection. In another aspect of the invention the method may comprise the treatment of a Grampositive bacterial infection.

[0052] The Gram-negative bacteria may be of a genus selected from Acinetobacter, Pseudomonas, Escherichia, Citrobacter, Enterobacter, Klebsiella, Proteus, Salmonella, Shigella, Serratia, Morganella, Bacteroides, Burkholderia, Campylobacter, Neisseria, Helicobacter, Haemophilus, Vibrio and Stenotrophomonas. In particular, a bacterial infection caused by a species of Acinetobacter, Pseudomonas, Escherichia, Citrobacter, Enterobacter, Klebsiella, Proteus, Salmonella, Shigella, Serratia, Morganella, Bacteroides, Burkholderia, Campylobacter, Neisseria, Helicobacter, Haemophilus, Vibrio and Stenotrophomonas is treatable by the methods disclosed herein.

[0053] Particular bacterial species for such treatment include Acinetobacter baumannii, Pseudonomas aeruginosa, E. coli, Klebsiella pneumoniae, Klebsiella oxytoca, Shigella spp., Salmonella spp., Campylobacter spp., Proteus mirabilis, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Citrobacter freundii, Citrobacter koseri, Neisseria gonorrhoeae, Helicobacter pylori, Haemophilus influenzae, Vibrio cholerae, Morganella morganii, Bacteroides fragilis, Burkholderia cepacia, and Stenotrophomonas maltophilia. The Gram-positive bacteria may be, for example, Staphylococcus aureus, Enterococcus faecium, Streptococcus pneumoniae and Clostridium botulinum.

[0054] As used herein, the term "subject" refers to an animal. In certain aspects, the animal is a mammal. A subject also refers to for example, primates (e.g. humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a human.

[0055] As used herein, the term "inhibition" or "inhibiting" refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process, or decrease in the viability, number or growth rate of a bacterial population.

[0056] As used herein, the term "treating" or "treatment" of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g. stabilisation of a discernible symptom), physiologically, (e.g. stabilisation of a physical parameter), or both. In yet another embodiment, "treating" or "treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder.

[0057] A method as herein defined comprising co-administration, e.g. concomitantly or in sequence, of a therapeutically effective amount of Annexin Al protein, or a variant thereof, and a second antibiotic as herein described.

[0058] The terms "co-administration" or "combined administration" or the like as utilized herein are meant to encompass administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. Fixed combinations are also within the scope of the present invention. The administration of a pharmaceutical combination of the invention results in a beneficial effect, e.g. a synergistic therapeutic effect, compared to a monotherapy applying only one of its pharmaceutically active ingredients.

[0059] Each component of a combination according to this invention may be administered separately, together, or in any combination thereof.

[0060] An "effective amount" of a combination of Annexin Al protein, or a variant thereof, and a second antibiotic refers to an amount effective to treat a bacterial infection in a subject, typically a human subject. The effective amount depends upon the sensitivity of the infecting bacterium to the chosen antibiotic. The skilled person can determine an effective amount of such combinations based on parameters of the subject to be treated, the infecting bacterium, and the combination to be used, which may include determining the MIC for the particular combination on the targeted bacterium. Typically, the bacterium to be treated is one that is resistant to conventional antibiotics.

[0061] The compositions of the invention also are useful in the treatment of patients suffering from or susceptible to brain infections, skin infections, pneumonia, sepsis, cystic fibrosis, wound, complicated diabetic foot, complicated intra-abdominal infections or complicated urinary tract infections and sexually transmitted diseases caused by Gram-negative or Gram-positive pathogens. The compositions of the invention also are useful in the conditions that are caused by a species of Acinetobacter, Pseudomonas, Escherichia, Klebsiella, Shigella, Salmonella, Campylobacter, Proteus, Enterobacter, Serratia, Citrobacter, Morganella, Neisseria, Helicobacter, Haemophilus, Vibrio, Bacteroides, Burkholderia, Stenotrophomonas, Staphylococcus, Enterococcus, Streptococcus or Clostridium. In particular, a bacterial infection caused by a species of Acinetobacter, Pseudomonas, Escherichia, Klebsiella, Shigella, Salmonella, Campylobacter, Proteus, Enterobacter, Serratia, Citrobacter, Morganella, Neisseria, Helicobacter, Haemophilus, Vibrio, Bacteroides, Burkholderia, Stenotrophomonas, Staphylococcus, Enterococcus, Streptococcus or Clostridium is treatable by methods herein.

[0062] Particular bacterial species for such treatment include Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Shigella spp., Salmonella spp., Campylobacter spp., Proteus mirabilis, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, Citrobacter freundii, Citrobacter koseri, Neisseria gonorrhoeae, Helicobacter pylori, Haemophilus influenzae, Vibrio cholerae, Morganella morganii, Bacteroides fragilis, Burkholderia cepacia, Stenotrophomonas maltophilia, Staphylococcus aureus, Enterococcus faecium, Streptococcus pneumoniae or Clostridium botulinum.

[0063] The language "pharmaceutical composition" includes preparations suitable for administration to mammals, e.g., humans. When the compositions of the present invention are administered as pharmaceuticals to mammals, e.g. humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0064] The phrase "pharmaceutically acceptable carrier" is art recognised and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering an Annexin Al protein and variants thereof as herein described to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminium hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. In some embodiments, a pharmaceutically acceptable carrier is sterilised before combination with the Annexin Al protein and variant thereof of the invention. In some embodiments, the pharmaceutical composition of the invention comprises Annexin Al protein as herein described and at least one pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition of the invention comprises Annexin Al protein as herein described and at least two pharmaceutically acceptable carriers or excipients.

[0065] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as preservatives and antioxidants can also be present in the compositions.

[0066] Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, a-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0067] Formulations of the present invention include those suitable for oral, nasal, inhalation, topical, transdermal, buccal, sublingual, rectal, vaginal and / or parenteral administration. Typically, compositions of the invention would be administered intravenously, in the form of a solution that is often isotonic, such as a saline or glucose solution. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of Annexin Al protein that produces a therapeutic effect. Generally, out of 100% w / w, this amount will range from about 1% to about 99% w / w of active ingredient, preferably from about 5% to about 70% w / w, most preferably from about 10% to about 30% w / w.

[0068] Pharmaceutical compositions of the invention suitable for parenteral administration comprise Annexin Al protein as herein described, in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0069] Examples of suitable aqueous and non-aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0070] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin.

[0071] Injectable forms may be made by forming microencapsule matrices of Annexin Al protein in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable formulations may also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0072] The formulations of the present invention may be given orally, parenterally, topically, or rectally. The formulations may be given in forms suitable for each administration route. For example, they may be administered in tablet or capsule form, by injection, e.g. intravenous injection, inhalation, eye lotion, ointment, suppository, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.

[0073] The phrases "parenteral administration" and "administered parenterally" used herein mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intra-capsular, intra-orbital, intra-cardiac, intradermal, intraperitoneal, trans-tracheal, subcutaneous, subcuticular, intraarticular, sub-capsular, subarachnoid, intra-spinal and intra-sternal injection and infusion.

[0074] The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" used herein mean the administration of Annexin Al protein as herein described other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

[0075] The Annexin Al protein or composition of the invention may be administered to humans and other animals for therapy by any suitable route of administration, including intramuscular injection, orally, nasally, inhaled as by, for example, a spray, rectally, intra-vaginally, parenterally, intra-cisternally and topically, as by powders, ointments or drops, including buccally and sublingually. In some embodiments, the Annexin Al protein or composition is administered by injection or infusion, and may be co-administered with another antibiotic. The other antibiotic may be administered by any appropriate route; in some embodiments, the other antibiotic is administered orally, and in other embodiments the other antibiotic is administered by injection or by infusion. When the Annexin Al protein or composition is co-administered with one or more additional antibiotics or other therapeutic agents, it is desirable that both are administered by the same route, they may optionally be admixed for administration by injection or by infusion, or they may be separately administered.

[0076] Actual dosage levels of the active ingredients, the Annexin Al protein and / or one or more additional antibiotics or other therapeutic agents, in the pharmaceutical composition of the invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0077] The selected dosage level will depend upon a variety of factors including the activity of the Annexin Al protein, the route of administration, the time of administration, the rate of excretion of the Annexin Al protein, the duration of the treatment, other drugs, compounds and / or materials used in combination with the Annexin Al protein, the age, sex, weight, condition, general health and prior medical history of the patient being treated, the genus, species and strain of bacterial pathogen causing the infection and like factors well known in the medical arts.

[0078] In general, a suitable daily dose of the Annexin Al protein will be that amount of the Annexin Al protein that is effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, intravenous and subcutaneous doses of the Annexin Al protein for a patient for the indicated antibacterial effects, will range from about 2 to about 100 mg per kg of body weight per day, more preferably from about 5 to about 100 mg per kg per day, and still more preferably from about 10 to about 50 mg per kg per day. An effective amount is the amount that treats a bacterial infection.

[0079] If desired, the effective daily dose of the Annexin Al protein may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms, or as continuous infusion.

[0080] While it is possible for Annexin Al protein to be administered alone, it is preferable to administer the Annexin Al protein as a pharmaceutical composition.

[0081] The invention is further illustrated by the following examples and figures, which should not be construed as limiting, in which:

[0082] Figure 1 illustrates antimicrobial activity of Annexin Al against penicillin-resistant S. pneumoniae,'

[0083] Figure 2 illustrates growth of Streptococcus pneumoniae (laboratory strain TIGR4) (Annexin Al (10 pg in 50 ml MQ water) added in the liquid cultures at 0 min. Experiment performed with three different liquid cultures of S. pneumoniae TIGR4 strain.);

[0084] Figure 3 provides the results of treatment with Annexin Al in Bacteremia-derived meningitis mouse model (Streptococcus pneumoniae TIGR4 strain);

[0085] Figure 4 illustrates the growth of Escherichia coli (strain ST131) (Annexin Al (10 pg in 50 ml MQ water) added in the liquid cultures at 0 min. Experiment performed with three different liquid cultures of A. coli ST131 strain.);

[0086] Figure 5 illustrates the results of cytotoxicity assays (LDH release) of Annexin Al in human hepatocytes (DMSO was used as positive control, untreated cells were the negative control- Annexin Al was given to the cells at the doses of 5 and 10 pg (in 50 ml seeding medium). Treatment was kept for twelve hours before measuring the degree of cell death.); Figure 6 illustrates the results of cytotoxicity assays (LDH release) of Annexin Al in human neurons (DMSO was used as positive control, untreated cells were the negative control- Annexin Al was given to the cells at the doses of 2.5 pg and 5 pg (in 50 ml seeding medium). Treatment was kept for twelve hours before measuring the degree of cell death.);

[0087] Figure 7 illustrates the clinical scoring of mice treated intravenously with Annexin Al over 5 consecutive days;

[0088] Figure 8 illustrates an assessment of the bacteriostatic or bactericidal effect of Annexin Al ;

[0089] Figure 9 illustrates the results of a cytotoxicity assessment of Annexin Al on SH- SY5Y-derived human neuronal cells;

[0090] Figure 10 illustrates the antimicrobial effects of Annexin Al on different bacterial strains;

[0091] Figure 11 illustrates the determination of the Minimum Inhibitory Concentration (MIC) of Annexin Al and albumin as a non-antimicrobial comparator;

[0092] Figure 12 illustrates the assessment of the bacterial growth and bacterial viability with Annexin Al ;

[0093] Figure 13 illustrates the results of cryo-electron microscopy on Streptococcus pneumoniae treated with Annexin Al ; and

[0094] Figure 14 illustrates an assessment of Anti-Microbial Resistance (AMR) in Streptococcus pneumoniae SP6 after the treatment with Annexin Al . The amino acid sequence of Annexin Al protein is provided below.

[0095] MAMVSEFLKQAWFIENEEQEYVQTVKSSKGGPGSAVSPYPTFNPSSDVAALH KAIMVKGVDEATIIDILTKRNNAQRQQIKAAYLQETGKPLDETLKKALTGHLE

[0096] EVVLALLKTPAQFDADELRAAMKGLGTDEDTLIEILASRTNKEIRDINRVYRE ELKRDLAKDITSDTSGDFRNALLSLAKGDRSEDFGVNEDLADSDARALYEAG ERRKGTDVNVFNTILTTRSYPQLRRVFQKYTKYSKHDMNKVLDLELKGDIEK CLTAIVKCATSKPAFFAEKLHQAMKGVGTRHKALIRIMVSRSEIDMNDIKAFY QKMYGISLCQAILDETKGDYEKILVALCGGN

[0097] Experimental

[0098] Cultivation of Streptococcus pneumoniae, Escherichia coli and Pseudomonas aeruginosa

[0099] Strains used in the study include Streptococcus pneumoniae SP6, Escherichia coli ST131 and Pseudomonas aeruginosa P476. SP6 was plated on blood agar, while P476 and ST131 was plated on agar plates overnight at 37°C, 5% CO2. Bacterial colonies from SP6 were collected and grown in Todd-Hewitt broth (0.5% yeast extract), while colonies from P476 and ST131 were collected and grown in Lysogeny broth (LB) at 37°C and harvested at OD620 = 0.25-0.3, aliquots stored at -80°C as a 25% glycerol solution.

[0100] Preparation of bacterial aliquots

[0101] Streptococcus pneumoniae SP6 was restreaked onto two blood agar plates and incubated overnight at 37°C with 5% CO2. Escherichia coli ST131 and Pseudomonas aeruginosa P476 were restreaked onto agar plates and incubated overnight at 37°C with 5% CO2. For liquid culture preparation, two 10 mL cultures of SP6 were prepared in THY broth using one blood agar plate for each culture. Optical density (OD) at 620 nm (OD62o) was monitored every 20 minutes until OD62o reached 0.25- 0.30, to ensure the bacteria were in the exponential growth phase. Bacterial cultures were mixed with glycerol (250 pL glycerol per mL of bacterial culture) and resuspended carefully to ensure a homogeneous suspension. The bacterial suspension was aliquoted into 1 mL Eppendorf tubes and stored at -80°C.

[0102] Culture of human cells

[0103] Neuroblastoma cells (SH-SY5Y, ATCC CRL-2266) were cultivated using Eagle’s Minimum Essential Medium (EMEM) and Ham’s F12 (Gibco) medium, 5% foetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% Penicillin- Streptomycin (P / S) (Thermo Fisher Scientific). SH-SY5Y-derived human neuronal cells were differentiated using 10 pM Retinoic Acid (RA) (Bio-Techne) for 12 days.

[0104] Culture of neuronal-like cells and in vitro cytotoxicity assays (infection + treatment)

[0105] SH-SY5Y-derived human neuronal cells were cultured as previously described. Briefly, cells were seeded in 12 well plates at a density of 100.000 cells / mL, differentiated for 12 days using differentiation media made of EMEM (ATCC) and F12 (Gibco) in 1 : 1 ratio, 2.5% foetal bovine serum (FBS) (Gibco), 1% penicillin / streptomycin (Gibco), and 10 mM retinoic acid (RA) (Bio-techne) and kept in incubator at 37°C with 5% CO2. Cytotoxicity assessment of Annexin Al protein towards neuronal cells

[0106] The lactate dehydrogenase (LDH) assay is a biochemical test used to measure the activity of the intracellular enzyme lactate dehydrogenase in various biological samples, as in our case is the SH-SY5Y-derived human neuronal cells. LDH catalyses the conversion of lactate to pyruvate, during which NADH is oxidized to NAD+. The amount of LDH is measured by the change in absorbance of NADH at 490 nm. The assay is performed as a colorimetric test: the presence of LDH in the supernatant indicates cell lysis and death. Each well of the cell plate is treated according to the experimental design. After treatment, the supernatant is collected to measure LDH levels. Higher LDH values indicate greater cellular cytotoxicity.

[0107] The day before performing LDH assay, neuronal-like cells were put in differentiation media without antibiotics and retinoic acid (EMEM and F12 in 1 : 1 ratio, 2.5% FBS), incubated Ih at 37°C, 5% CO2. The seeding media was then removed from each well and cells were gently washed with ImL pre-heated PBS for 5 min. PBS was removed and ImL seeding media was added in each well. Cells were incubated Ihr at 37°C, 5% CO2. The negative control well was tested without adding any Annexin Al protein, to assess the cytotoxicity of the neural cell line alone. For the positive control, Img / mL of Cellular Lysate Buffer, provided by the LDH kit, was added to the sample well. A titration of Annexin Al protein was performed during the experiment: cells were challenged with respectively with 10 pg / mL, 25 pg / mL, 50 pg / mL and 100 pg / mL of Annexin Al protein. Subsequently, the plate was incubated at 37°C, 5% CO2. After 2 hours of incubation, 50 pL of each sample medium (spontaneous LDH activity, maximum LDH activity and Annexin Al protein treated LDH activity) was transferred to a 96 well flat bottom plate in triplicate wells. 50 pL of Reaction Mixture provided by the LDH kit was added to each sample well, and the plate was incubated at room temperature for 30 min, protected from light. 50 pL of Stop Solution was added to each sample well, then it was gently mixed by tapping. It is important to break any bubbles present in wells with a syringe needle and / or by centrifugation before the plate reading. Absorbance was measured at 490 nm and 680 nm. In order to determine LDH activity, the 680 nm absorbance value (background) was subtracted from the 490 nm absorbance before calculation of % cytotoxicity. Data analysis was performed using GraphPad software.

[0108] Infection of SH-SY5Y-derived human neuronal cells and treatment with Annexin Al protein

[0109] The day before the bacterial infection, neuronal-like cells were put in differentiation media without antibiotics (EMEM and F12 in 1 : 1 ratio, 2.5% FBS, lOmM RA). The day of the experiment, the medium was discarded, cells were washed twice with PBS and put in EMEM and F12 in 1 : 1 ratio, 2.5% FBS, incubated Ih at 37°C, 5% CO2. For the infection experiments, 1 mL aliquots of Streptococcus pneumoniae SP6, Pseudomonas aeruginosa P476 and Escherichia coli ST131 strain (OD620 of 0.25- 0.30) were used; neuronal-like cells were then infected with Multiplicity of Infection (MOI)=50 for all the strains. Bacterial cells were washed with PBS, resuspended in cell culture medium without the addition of P / S and RA, and subsequently 0.5 mL of MOI 50 medium was added to each well. After the bacterial addition, plates were centrifuged at 50 RPF for 10 min. The negative control sample was run without the addition of either bacteria and Annexin Al protein, to check the cytotoxicity of the neural cell line only. The positive control was run adding to the sample well Img / mL of Cellular Lysate Buffer, supplied by the LDH kit. Another control was run performing only the infection with the selected MOI of 50, without the treatment afterwards. Two wells were designed to be infected with the bacteria for 90 min, before adding Annexin Al protein to each well, to reach respectively a final concentration of 10 pg / mL. Another important control was the addition of bacteria resuspended in 10 pg / mL of Annexin Al protein in PBS.

[0110] After 5 hours incubation, 50 mL of each sample (=well) were transferred into a 96- well flat-bottom plate, in technical triplicates. Three biological samples were analysed. During the assay, supernatant aliquots were kept at 4°C, neuronal-like cells were kept at 37°C with 5% CO2. CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen, Cat. C20300 and C20301) was used to measure the released lactase dehydrogenase (LDH), as assessment of cytotoxicity. Absorbance was measured with Multiskan FC microplate reader (Thermo Fisher Scientific) at 450 nm and 620 nm; to determine LDH activity, 620 nm absorbance value (background from the instrument) was subtracted to 450 nm absorbance value.

[0111] CFU count for MIC determination of Annexin Al protein

[0112] 200 pL of bacterial suspension was aliquoted into Eppendorf tubes. Aliquots were centrifuged at 10,000 rpm for 3 minutes to pellet the bacteria. The supernatant was removed, and the bacterial pellet was resuspended in 200 pL of the appropriate solutions: PBS (negative control), 1 pg / mL of penicillin in PBS or 2 pg / mL carbapenem in PBS, and a titration of Annexin Al protein, respectively: 10 pg / mL, 5 pg / mL, 2.5 pg / mL and 1.25 pg / mL of Annexin Al protein in PBS. Serial dilutions were prepared using a 96-well plate. Dilutions ranged from 101to 106. Three drops of 10 pL from each dilution were plated onto the appropriate agar plates (blood agar for SP6 and standard agar for P476 and ST 131). The plates were left to dry at room temperature with the lids off. Once dried, the plates were placed upside down in a 37°C incubator with 5% CO2. and left to grow overnight. The experiment was performed in triplicates. The next day, colonies were counted, and the colony-forming units (CFU) per mL were calculated, multiplying by the dilution factor.

[0113] CFU count with albumin treatment

[0114] 200 pL of bacterial aliquots were centrifuged at 10,000 rpm for 3 minutes and resuspended in 200 pL of the appropriate solutions: bacterial medium (either THY or LB medium depending on the bacterial strain, as negative control), 1 pg / mL of penicillin in PBS or 2 pg / mL carbapenem in PBS, and 5 pg / mL and 10 pg / mL of albumin resuspended in PBS. Serial dilutions were prepared using a 96-well plate. Dilutions ranged from 101to 106. Three drops of 10 pL from each dilution were plated onto the appropriate agar plates (blood agar for SP6 and standard agar for P476 and ST131). The plates were left to dry at room temperature with the lids off. Once dried, the plates were placed upside down in a 37°C incubator with 5% CO2, and left to grow overnight. The experiment was performed in triplicates.

[0115] The next day, colonies were counted, and the colony-forming units (CFU) per mL were calculated, multiplying by the dilution factor. Bacterial growth curve

[0116] Streptococcus pneumoniae SP6, Escherichia coli ST 131, and Pseudomonas aeruginosa P476 were individually cultured in appropriate growth media, as previously explained.

[0117] For growth curve measurements, 180 pL of each diluted bacterial culture was added to separate wells of a clear 96-well microplate. The plate was placed in a Multiskan FC microplate reader (Thermo Fisher Scientific) preheated to 37°C. Absorbance readings at OD62o were taken every 10 minutes for a total of 180 minutes (18 measurements) to monitor bacterial growth.

[0118] Before starting the experiment, each bacterial aliquot was plated as previously explained in the CFU count paragraph, to assess the CFU / mL bacterial value at the initial time point of the experiment.

[0119] After the initial measurements, Annexin Al protein was added to the wells at a final concentration of 10 pg / mL, 50 pg / mL, 200 pg / mL. A separate negative control well was prepared without the addition of Annexin Al protein. Additionally, control wells were treated with 1 pg / mL of penicillin in PBS for the penicillin-resistant S. pneumoniae SP6 and 2 pg / mL carbapenem in PBS for carbapenem-resistant E. coli ST131 and P. aeruginosa P476. The microplate was placed back into the Multiskan FC microplate reader, and growth curve measurements were continued under the same conditions for an additional 180 minutes (18 measurements). The absorbance readings obtained at OD62o were plotted against time to generate growth curves for each bacterial strain under different experimental conditions. The effect of Annexin Al protein on bacterial growth was assessed by comparing the growth curves of treated wells to those of untreated controls and antibiotic-treated controls.

[0120] At the end of the experiment, the bacterial suspension was plated according to the method described in the CFU count section. This was done to determine the CFU / mL at the final time point, allowing us to assess whether the OD values accurately reflect bacterial viability.

[0121] Cryo-Electron Microscopy

[0122] Streptococcus pneumoniae SP6 cultures were grown to OD 620 of 0.3-0.35 and divided into two groups: untreated control and treated samples. The untreated control samples were resuspended in phosp hate-buffered saline (PBS). The treated samples were resuspended in a solution of PBS containing 10 pg / mL of Annexin Al. Both sets of samples were incubated at 37°C, 5% CO2 for 45 minutes to allow interaction between the bacteria and Annexin Al .

[0123] After incubation, both untreated and treated samples were fixed using 4% formaldehyde (FA) for 30 minutes at room temperature. Following fixation, the samples were embedded in 10% gelatin. The gelatin-embedded samples were cryoprotected by immersion in 2.3 M sucrose overnight at 4°C. Subsequently, the cryoprotected samples were mounted on cryo pins and rapidly frozen in liquid nitrogen. The frozen samples were cryosectioned into ultrathin sections (50-70 nm) using an ultramicrotome equipped with a cryo-chamber at -120°C. The cryosections were transferred onto formvar-coated nickel grids using a solution of methyl cellulose and 2.3 M sucrose (1 : 1 mixture).

[0124] The grids were blocked with 1% bovine serum albumin (BSA) in PBS for 30 minutes at room temperature to prevent nonspecific binding. The blocked grids were incubated Mouse Annexin Al Antibody, Monoclonal Mouse IgGl Clone # 686106, diluted 1 : 100 in PBS containing 1% BSA, for 1 hour at room temperature. After primary antibody incubation, the grids were washed 5 times with PBS containing 0.1% BSA to remove unbound primary antibody. The grids were then incubated with gold- conjugated Protein- A, specific to mouse antibodies, diluted 1 : 100 in PBS containing 1% BSA, for 1 hour at room temperature. Following secondary antibody incubation, the grids were washed extensively with PBS containing 0.1% BSA and then with distilled water to remove any residual salts. The labelled grids were contrasted using a solution of 2% uranyl acetate in 0.15 M oxalic acid for 5 minutes, followed by embedding in a thin layer of methyl cellulose. The grids were examined using a transmission electron microscope (TEM) operated at 80 kV. Images were acquired to evaluate the localization and distribution of the immunogold labelling within the bacterial cells.

[0125] As a control sample, untreated SP6 samples were processed in parallel without the addition of Annexin Al. These control samples were subjected to the same immunolabelling protocol to ensure specificity of the primary and secondary antibodies and the absence of nonspecific binding.

[0126] Antimicrobial Resistance Acquisition Test

[0127] Streptococcus pneumoniae SP6 (200 pL bacterial aliquots), passage 1, were centrifuged at 10,000 rpm for 3 minutes and resuspended in 200 pL of the appropriate solutions: THY bacterial medium, and 5 pg / mL of Annexin Al protein resuspended in PBS. This concentration corresponds to half the minimum inhibitory concentration (MIC) for SP6, ensuring bacterial contact with the protein without causing complete bacterial death.

[0128] Serial dilutions were prepared using a 96-well plate, ranging from 101to 106. Three drops of 10 pL from each dilution were plated onto blood agar plates. The plates were allowed to dry at room temperature with the lids off. Once dried, the plates were incubated upside down at 37°C with 5% CO2 overnight.

[0129] The next day, 200 colonies from each condition were counted and resuspended in 2 mL THY medium. Optical density (OD) at 620 nm (OD620) was monitored every 30 minutes for a total of 4 hours to observe the bacterial growth rate. The aim was to determine whether the presence of the Annexin Al protein led to resistance acquisition, indicated by faster or consistent growth rates.

[0130] At the end of each growth curve, the experiment was restarted using 200 pL of the bacterial liquid culture as the starting point. This loop was performed five times to test for resistance acquisition. The experiment was conducted in triplicate. Examples

[0131] Example 1

[0132] Antimicrobial activity of Annexin Al against penicillin-resistant S. pneumoniae

[0133] Referring to Figure 1, growth (measured in CFU / ml) of the penicillin-resistant S. pneumoniae strain SP6 was assessed on blood agar plates without any treatment (no treatment), or treated with penicillin, with purified Annexin Al at doses of 5 or 10 pg (in 50 pl MQ water spread onto blood-agar plates). Bacterial growth in the presence of Annexin Al was compared with growth after treatment with penicillin. ** = p<0.01, *** = p<0.001, n.s. = non-significant.

[0134] Example 2

[0135] Results of treatment with Annexin Al in Bacteremia-derived meningitis mouse model (Streptococcus pneumoniae TIGR4 strain)

[0136] Referring to Figure 3, Five C57BL / 6 male mice per group. Intravenous (iv) injection with 10A8 CFU / mouse of Streptococcus pneumoniae TIGR4 strain. Annexin Al (5 or 10 pg in 100 pl sterile PBS) was administered iv two hours after bacterial infection. Infection experiment lasted for three consecutive days. Brains collected at the time of euthanasia and used for bacterial CFU count (all mice were perfused after sacrifice). Time of euthanasia

[0137] • Untreated mice: End day 1 (severe sickness, humane end-point reached).

[0138] • Mice treated with Annexin Al : End Day 3 (no symptoms of disease).

[0139] Bacteremia levels Untreated mice: 1.1 x 10A9 CFU / ml average value (severe bacteremia)

[0140] Mice treated with Annexin Al 5 pg: 2.2 x 10A2 CFU / ml average value (mild bacteremia)

[0141] Mice treated with Annexin Al 5 pg: 1.3 x 10A2 CFU / ml average value (mild bacteremia); notably, two mice had no bacteria in the blood.

[0142] Example 3

[0143] Clinical scoring of mice treated IV with Annexin Al over 5 consecutive days

[0144] Referring to Figure 7, before testing the efficacy of Annexin Al as an antibiotic in vivo, we first tested the absence of unwanted side effects of Annexin Al in mice without infection.

[0145] Five C57BL / 6 wild-type male mice were treated intravenously (IV) with 10 pg Annexin Al (in 100 pl sterile PBS) and monitored for 5 consecutive days; the health status of the animals was checked twice a day.

[0146] The clinical scoring was assessed according to the health status assessment of rodents at the Karolinska Institutet (0.0 = healthy mouse; 0.5-0.6 = sick mouse to be euthanized).

[0147] All mice remained healthy without signs of side effects for five consecutive days after

[0148] IV treatment with Annexin Al . Example 4

[0149] Cytotoxicity and Antimicrobial Efficacy of Annexin Al protein

[0150] Cytotoxicity levels of the Annexin Al protein were assessed using an LDH assay on SH-SY5Y-derived human neuronal cells.

[0151] Referring to Figure 9, the cells were seeded and differentiated for 12 days in differentiation media, maintained in an incubator at 37°C with 5% CO2. The experimental conditions included treatment with medium only (negative control), cellular lysate buffer (positive control provided by the LDH kit), and a titration of Annexin Al protein (from 10 pg / mL to 100 pg / mL, as shown in the figure). Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) activity, where the absorbance at 680 nm (background) was subtracted from the absorbance at 490 nm before calculating the percentage of cytotoxicity. Data analysis was conducted using GraphPad software. The experiments included three biological and three technical replicates.

[0152] As illustrated in Figures 5, 6 and 9, Annexin Al protein did not exhibit cytotoxic effects on human hepatocytes and human neurons. This result aligns with expectations, given that Annexin Al is a human protein and is not anticipated to harm hepatocytes and neural cells.

[0153] Following this, a colony-forming unit (CFU) assay was conducted. Penicillin-resistant Streptococcus pneumoniae SP6, carbapenem-resistant Pseudomonas aeruginosa, and carbapenem-resistant Escherichia coli ST131 were resuspended in PBS containing various concentrations of Annexin Al protein. Referring to Figure 10, (A) CFU count of the penicillin-resistant Streptococcus pneumoniae SP6 in different experimental conditions, which included: bacteria resuspended in THY medium (medium only), 1 pg / mL penicillin, or varying concentrations of Annexin Al protein (ranging from 10 pg / mL to 100 pg / mL in PBS), as indicated on the x-axis. (B) CFU count of carbapenem-resistant Pseudomonas aeruginosa P476 in different experimental conditions. Bacteria were exposed to LB medium, 2 pg / mL carbapenem, 25 pg / mL Annexin Al protein in PBS. (C) CFU count of carbapenem-resistant Escherichia coli ST 131, treated with LB medium, 2 pg / mL carbapenem, 25 pg / mL Annexin Al protein. Three biological and two technical replicates were performed.

[0154] As shown in Figure 10, Annexin Al protein demonstrated potent antimicrobial activity, completely eliminating bacterial colonies. This was evidenced by the absence of CFUs after treatment, with CFU / mL values falling below the detection limit of the plating assay, indicating high efficacy.

[0155] To further elucidate the Minimum Inhibitory Concentration (MIC) of Annexin Al protein for each bacterial strain, lower concentrations were tested.

[0156] Referring to Figure 11, (A) MIC of Annexin Al protein for three different bacterial strains: penicillin-resistant Streptococcus pneumoniae SP6, carbapenem-resistant Escherichia coli ST131 and carbapenem-resistant Pseudomonas aeruginosa P476. Bacterial aliquots were resuspended in a series of Annexin Al protein dilutions. The detection limit of this technique is 103CFU / mL. The experiment included three technical replicates. Statistical analysis was performed using GraphPad software. **** indicates a p-value < 0.0001. (B) The antibiotic effect of albumin was tested on three bacterial strains: penicillin-resistant Streptococcus pneumoniae SP6, carbapenem-resistant Escherichia coli ST131 and carbapenem-resistant Pseudomonas aeruginosa P476. The experiment was performed in three technical replicates. Statistical analysis was conducted using GraphPad software, "ns" indicates a non- statistically significant difference.

[0157] As shown in Figure 11 A, the MIC values were determined to be 10 pg / mL for Streptococcus pneumoniae and Escherichia coli, while Pseudomonas aeruginosa required an even lower concentration of 5 pg / mL. These findings underscore the potent antimicrobial activity of Annexin Al protein, with low MIC values indicating significant efficacy against the tested bacterial strains.

[0158] As a control, a CFU assay was performed on Streptococcus pneumoniae using albumin (Figure 1 IB). Albumin did not affect bacterial viability, with no significant difference in CFU / mL between albumin-treated and untreated bacteria.

[0159] These results highlight the effectiveness of Annexin Al protein as an antimicrobial agent, demonstrating its potential to combat antibiotic-resistant bacterial strains without cytotoxic effects on human neuronal cells. Example 5

[0160] Assessment of the Effect of Annexin Al protein on bacteria

[0161] To determine whether Annexin Al protein exhibits bactericidal or bacteriostatic effects, we monitored bacterial growth over time using a microplate reader at OD 620 nm and 37°C. Optical density measurements were taken before and after the addition of the Annexin Al protein.

[0162] To confirm what seemed like a bactericidal effect from the results of Figure 10, and to evaluate the effect of a higher concentration of Annexin Al protein, growth curves were generated for the penicillin-resistant Streptococcus pneumoniae SP6 strain as shown in Figure 8. (A) Bacteria were exposed to 50 pg / ml of Annexin Al protein in PBS at two different time points: 3 hours and 6 hours. The grey circle indicates the specific time point at which the Annexin Al protein, penicillin or medium was added. (B) Bacteria were exposed to 200 pg / ml of Annexin Al protein in PBS at one single time point. The grey circle indicates the specific time point (3 hours) at which the Annexin Al protein, penicillin or medium was added. Statistical analysis was performed using one-way ANOVA, with Bonferroni correction for the multiple comparison test. *** indicates a p-value < 0.001.

[0163] Referring to Figure 8, the optical density levels remained unchanged and steady after the addition of Annexin Al protein, initially suggesting a potential bacteriostatic effect regardless of the concentration tested. An evaluation of bacterial viability was then performed for further confirmation. To assess bacterial viability, growth curves were generated for three bacterial strains: carbapenem-resistant Escherichia coli ST 131, penicillin-resistant Streptococcus pneumoniae SP6, and penicillin-sensitive Streptococcus pneumoniae TIGR4. Bacteria were exposed to the MIC concentrations of Annexin Al protein. Bacterial viability was assessed at the beginning and at the end of the experiment by plating the bacterial suspension onto agar plates. The detection limit of the plating technique is 103CFU / mL. Each condition included three technical replicates. Statistical analysis was performed using one-way ANOVA, with Bonferroni correction for the multiple comparison test. *** indicates a p-value < 0.001.

[0164] Referring to the first and second panels of Figure 12, the optical density levels remained unchanged and steady after the addition of Annexin Al protein, initially suggesting a potential bacteriostatic effect in line with Figure 8. However, the plating of bacterial liquid cultures in the wells of the 96-well-plate revealed that no viable bacteria were present in the liquid culture, indicating that the steady OD values were due to the presence of dead bacterial cells. These dead cells contributed to the optical density reading, even though they were not viable, as confirmed by the plating results immediately after the measurements.

[0165] In contrast, Streptococcus pneumoniae TIGR4 penicillin- sensitive strain challenged with penicillin results in bacterial lysis and a sudden drop in OD values and bacterial CFU (Figure 12- panel 3).

[0166] This finding suggests a potent bactericidal effect of Annexin Al protein on the tested bacteria. The data imply that Annexin Al effectively kills the bacteria, rather than only inhibiting their growth, thus demonstrating its potential as a powerful antimicrobial agent.

[0167] Example 6

[0168] Fixative effect on bacteria as mechanism of action

[0169] To elucidate the mechanism by which Annexin Al protein exerts bactericidal effects, we conducted cryo-electron microscopy staining. Streptococcus pneumoniae was treated with Annexin Al labelled with colloidal gold particles.

[0170] Referring to Figure 13, the top panel shows the untreated control cells, revealing the typical morphology of Streptococcus pneumoniae SP6. In contrast, the bottom panel displays cells treated with 10 pg / mL Annexin Al protein (MIC concentration), highlighted with immunogold particles (black signal) indicating the presence of Annexin Al protein. Additionally, the light white signal surrounding the cellular structure represents the overexpressed capsule. The comparison between the panels elucidates the morphological changes and Annexin Al localization within the treated bacterial cells.

[0171] As shown in Figure 13, significant morphological differences were observed between the treated (bacteria incubated with Annexin Al for 45 minutes before fixation) and untreated samples (negative control, only fixed bacteria without treatment).

[0172] In the treated samples, the bacterial capsule appeared enlarged, and the cellular wall was faint and indistinct. In contrast, the control samples exhibited a dark, well-defined cellular wall with a smaller capsule. The enlargement of the capsule in Streptococcus pneumoniae is a well-documented adaptive response to external threats, enhancing the bacterium's ability to resist hostile conditions and immune responses. This response often involves increased capsule thickness and a more robust defence against environmental stressors and antimicrobial agents.

[0173] Notably, no lysed bacteria were observed in the treated samples; all bacteria appeared intact, despite the altered morphology. This observation led us to hypothesize that Annexin Al functions via a "fixative-like mechanism," interfering with the bacterial cell wall specifically, the peptidoglycan — resulting in bacterial death without causing cell lysis.

[0174] This finding is crucial as it suggests that Annexin Al protein kills bacteria without releasing intracellular toxins, thereby minimizing the potential for a strong immune response and cytotoxic effects. Further analyses are necessary to confirm this hypothesis and to fully understand the implications of this mechanism. If validated, Annexin Al could represent a novel approach to antimicrobial therapy, effectively eliminating bacteria while reducing the risk of adverse immune reactions.

[0175] Example 7

[0176] Development of Anti-Microbial Resistance

[0177] A significant aspect of this study was the assessment of antimicrobial resistance (AMR) development. The bacterial strain selected for this experiment was Streptococcus pneumoniae SP6, which underwent serial passaging in either medium alone, or 5 pg / mL Annexin Al protein in PBS.

[0178] Referring to Figure 14, AMR was tested under two different conditions: Streptococcus pneumoniae SP6 was resuspended in Todd-Hewitt Broth w / 2% Yeast Extract (THY) medium as a negative control, and treatment with 5 pg / mL Annexin Al protein in PBS. The selected concentration of the protein corresponds to half of the MIC for SP6, ensuring sub-inhibitory exposure. The growth of SP6 was monitored by measuring OD620 over a period of 4 hours. To evaluate potential changes in antimicrobial resistance, SP6 was subjected to serial passaging, with cultures transferred to fresh media and treatments for a total of 5 passages. One biological replicate was performed.

[0179] As illustrated in Figure 14, the control sample maintained a consistent growth curve throughout the experiment. In contrast, the samples treated with Annexin Al protein, at half the MIC value, exhibited substantially slower growth rates.

[0180] This finding suggests that the bacteria did not develop resistance to the treatments, as evidenced by the absence of accelerated growth which would indicate adaptation. Instead, the reduced growth rate indicates a continued susceptibility to the sub-MIC concentrations of Annexin Al protein. This outcome supports the potential efficacy of Annexin Al in inhibiting bacterial growth without developing resistance, offering a promising strategy for combating AMR.

Claims

Claims1. Annexin Al protein, and variants thereof, for use in the treatment of a microbial disorder.

2. Annexin Al protein according to claim 1 wherein the microbial disorder comprises disorders caused by bacteria and other microbes, such as, parasites (e.g. malaria), viruses (e.g. HIV) and fungi (e.g. Candida).

3. Annexin Al protein according to any one of the preceding claims wherein the microbial disorder comprises a bacterial disorder.

4. Annexin Al protein according to claim 3 wherein the bacterial disorder is caused by Gram-negative bacteria.

5. Annexin Al protein according to claim 3 wherein the bacterial disorder is caused by Gram-positive bacteria.

6. Annexin Al protein according to any one of the preceding claims 1 wherein the Annexin Al protein is microbicidal or microbiostatic.

7. Annexin Al protein according to claim 6 wherein the Annexin Al protein is bactericidal and / or bacteriostatic.

8. Annexin Al protein according to any one of the preceding claims wherein the variants of the Annexin Al protein may possess substitutions, additions, deletions and other modifications and lead to derived proteins.

9. Annexin Al protein according to claim 8 wherein the variants possess at least about 50% identity (homology) to the reference to Annexin Al protein.

10. Annexin Al protein according to claim 3 wherein the bacterial disorder is caused by multi-drug resistant Gram-positive bacteria or multi-drug resistant Gramnegative bacteria.

11. Annexin Al protein according to claim 3 wherein the bacterial disorder is caused by bacteria that exhibit resistance to beta-lactam antibiotics.

12. Annexin Al protein according to claim 3 wherein the bacterial disorder is caused by Gram-negative bacteria, including, but not limited to, Acinetobacter e.g. Acinetobacter baumannii, Pseudomonas e.g. Pseudomonas aeruginosa, Enterobacteriaceae (e.g. Escherichia coli (E. colt), Klebsiella pneumoniae, Shigella spp., Salmonella spp.), Campylobacter spp., Neisseria e.g. Neisseria gonorrhoeae, Helicobacter e.g. Helicobacter pylori, Haemophilus e.g. Haemophilus influenzae, and Vibrio e.g. Vibrio cholerae.

13. Annexin Al protein according to claim 3 wherein the bacterial disorder is caused by Gram-positive bacteria, including, but not limited to, Staphylococcus, Streptococcus, Enterococcus, and Clostridium.

14. Annexin Al protein according to claim 11 wherein the bacterial disorder is caused by Gram-negative bacteria, including, but not limited to, Acinetobacter e.g. Acinetobacter baumannii, Pseudomonas e.g. Pseudomonas aeruginosa, Enterobacteriaceae (e.g. Escherichia coli (E. coli), Klebsiella pneumoniae, Shigella spp., Salmonella spp., Campylobacter spp., Proteus, Enterobacter, Serratia, and Citrobactef), Neisseria e.g. Neisseria gonorrhoeae, Helicobacter e.g. Helicobacter pylori, Haemophilus e.g. Haemophilus influenzae, Vibrio e.g. Vibrio cholerae, , nonfermenting bacteria, including Burkholderia, Moraxella and Stenotrophomonas, , as well as anaerobic bacteria, such as Bacteroides fragilis or Bacteroides thetaiotaomicron,' or Gram-positive bacteria, including, but not limited to, Staphylococcus e.g. Staphylococcus aureus, Enterococcus e.g. Enterococcus faecium, Streptococcus e.g. Streptococcus pneumoniae, and Clostridium e.g. Clostridium botulinum, inclusive of drug-resistant and multi-drug resistant strains.

15. Annexin Al protein according to claim 11 wherein the bacterial disorder is caused by a pathogen appearing on the World Health Organisation (WHO) list of priority pathogens: WHO updates list of drug-resistant bacteria most threatening to human health.

16. Annexin Al protein according to claim 11 wherein the bacterial disorder is caused by Gram negative strains, such as, E. coli, Pseudomonas aeruginosa,' andGram positive strains, such as, Streptococcus pneumoniae & Staphylococcus aureus.

17. A pharmaceutical composition comprising Annexin Al protein, and variants thereof, admixed with at least one pharmaceutically acceptable carrier or excipient, for use in the treatment of a microbial disorder.

18. A pharmaceutical composition according to claim 17 wherein the microbial disorder comprises disorders caused by bacteria and other microbes, such as, parasites (e.g. Plasmodium), viruses (e.g. HIV) and fungi (e.g. Candida).

19. A pharmaceutical composition according to any one of claims 17 or 18 wherein the microbial disorder comprises a bacterial disorder.

20. A pharmaceutical composition according to claim 19 wherein the bacterial disorder is caused by Gram-negative bacteria.

21. A pharmaceutical composition according to claim 19 wherein the bacterial disorder is caused by Gram-positive bacteria.

22. A pharmaceutical composition according to any one of claims 17 to 21 wherein the Annexin Al protein is microbicidal or microbiostatic.

23. A pharmaceutical composition according to claim 22 wherein the Annexin Al protein is bactericidal and / or bacteriostatic.

24. A pharmaceutical composition according to any one of claims 17 to 23 wherein the composition comprises two or more such carriers or excipients.

25. A pharmaceutical composition according to any one of claims 17 to 24 wherein the composition includes Annexin Al protein in combination with one or more additional antibiotics or other therapeutic agents.

26. A pharmaceutical composition according to claim 25 wherein the other antibiotic may be selected from one or more of the antibiotic classes aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins glycopeptides, lincosamides, lipopeptide, macrolides, monobactams, nitrofiirans, oxazolidinones, penicillins, polypeptides, quinolones, sulfonamides, tetracyclines and drugs against mycobacteria.

27. A method for treating a subject having a microbial infection, said method comprising administering to the subject in need thereof an antimicrobially effective amount of Annexin Al protein.

28. A method according to claim 27 wherein the microbial disorder comprises disorders caused by bacteria and other microbes, such as, parasites (e.g. malaria), viruses (e.g. HIV) and fungi (e.g. Candida).

29. A method according to any one of claims 27 or 28 wherein the microbial disorder comprises a bacterial disorder.

30. A method according to claim 29 wherein the bacterial disorder is caused byGram-negative bacteria.

31. A method according to claim 29 wherein the bacterial disorder is caused byGram-positive bacteria.

32. A method according to claims 27 to 31 wherein the Annexin Al protein is microbicidal or microbiostatic.

33. A method according to claim 32 wherein the Annexin Al protein is bactericidal and / or bacteriostatic.

34. A method according to any one of claims 27 to 33 wherein the composition comprises two or more such carriers or excipients.

35. A method according to any one of claims 27 to 34 wherein the composition includes Annexin Al protein in combination with one or more additional antibiotics or other therapeutic agents.

36. A method according to claim 35 wherein the other antibiotic may be selected from one or more of the antibiotic classes aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins glycopeptides, lincosamides, lipopeptide, macrolides, monobactams, nitrofiirans, oxazolidinones, penicillins, polypeptides, quinolones, sulfonamides, tetracyclines and drugs against mycobacteria.

37. A method according to any one of claims 27 to 36 wherein the method comprises administering a pharmaceutical composition, i.e. optionally in combination with a pharmaceutically acceptable carrier.

38. A method according to any one of claims 27 to 37 wherein the subject is a human.

39. A method according to any one of claims 27 to 38 wherein the microbial infection is a Gram-negative bacterial infection.

40. A method according to claim 39 wherein the Gram-negative bacterial infection is of a genus selected from Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Proteus, Salmonella, Serratia, Pseudomonas, Acinetobacter, Bacteroides, Burkholderia, Campylobacter, Neisseria, and Stenotrophomonas.

41. A method according to any one of claims 27 to 40 wherein the microbial infection is a Gram-positive bacterial infection.

42. A method according to claim 41 wherein the Gram-positive bacterial infection is of a genus selected from Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus or Streptococcus pneumoniae.

43. A method according to claim 29 wherein the bacterial disorder is caused by multi-drug resistant Gram-positive bacteria or multi-drug resistant Gram-negative bacteria.

44. A method according to claim 29 wherein the bacterial disorder is caused by bacteria that exhibit resistance to beta-lactam antibiotics.

45. A method according to claim 29 wherein the bacterial disorder is caused by Gram-negative bacteria, including, but not limited to, Acinetobacter e.g. Acinetobacter baumannii, Pseudomonas e.g. Pseudomonas aeruginosa, Enterobacteriaceae (e.g. Escherichia coli (E. coli), Klebsiella pneumoniae, Shigella spp., Salmonella spp.), Campylobacter spp., Neisseria e.g. Neisseria gonorrhoeae, Helicobacter e.g. Helicobacter pylori, Haemophilus e.g. Haemophilus influenzae, and Vibrio e.g. Vibrio cholerae.

46. A method according to claim 29 wherein the bacterial disorder is caused by Gram-positive bacteria, including, but not limited to, Staphylococcus, Streptococcus, Enterococcus, and Clostridium.

47. A method according to claim 29 wherein the bacterial disorder is caused by Gram-negative bacteria, including, but not limited to, Acinetobacter e.g. Acinetobacter baumannii, Pseudomonas e.g. Pseudomonas aeruginosa, Enterobacteriaceae (e.g. Escherichia coli (E. coll), Klebsiella pneumoniae, Shigella spp., Salmonella spp., Campylobacter spp., Proteus, Enterobacter, Serratia, and Citrobacter), Neisseria e.g. Neisseria gonorrhoeae, Helicobacter e.g. Helicobacter pylori, Haemophilus e.g. Haemophilus influenzae, Vibrio e.g. Vibrio cholerae, nonfermenting bacteria, including Burkholderia, Moraxella and Stenotrophomonas, as well as anaerobic bacteria, such as Bacteroides fragilis or Bacteroidesthetaiotaomicron,' or Gram-positive bacteria, including, but not limited to, Staphylococcus e.g. Staphylococcus aureus, Enterococcus e.g. Enterococcus faecium, Streptococcus e.g. Streptococcus pneumoniae, and Clostridium e.g. Clostridium botulinum, inclusive of drug-resistant and multi-drug resistant strains.

48. A method according to claim 29 wherein the bacterial disorder is caused by a pathogen appearing on the World Health Organisation (WHO) list of priority pathogens : WHO updates list of drug-resistant bacteria most threatening to human health.

49. A method according to claim 29 wherein the bacterial disorder is caused by Gram negative strains, such as, E. coli, Pseudomonas aeruginosa,' and Gram positive strains, such as, Streptococcus pneumoniae & Staphylococcus aureus.

50. Annexin Al protein, pharmaceutical composition, method, as herein described with reference to the accompanying examples.

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